Download GBA deficiency promotes SNCA/α-synuclein accumulation through

GBA deficiency promotes SNCA/α-synuclein accumulation
through autophagic inhibition by inactivated PPP2A
Supplemental material
Figure S1. Efficient infection upon GBA knockdown. Neurons infected with LV-GFP-shGba #6
and LV-GFP-sh-con for 3 days, and visualized by treatment with an antibody against
TUBB3/tubulin III and staining with DAPI. Scale bar: 25 μm.
Figure S2. The accumulation of the substrate GlcCer upon GBA knockdown. (A) Samples
were imaged at the same exposure to allow direct comparisons. (B) GlcCer staining intensity
was calculated using ImageJ software. Increased GlcCer in cells infected with LV-GFP-shGBA
#9848-1. Data were analyzed for 10 randomly selected fields of view from 3 independent
experiments. **, P< 0.01 vs. LV-GFP-sh-con group. Scale bar: 10 μm.
Figure S3. C2-ceramide upregulates BECN1 level in GBA-deficient cortical neurons. (A, B)
BECN1 protein level was reduced in cortical neurons infected with LV-GFP-shGba #6, which
was restored by treatment with exogenous C2 (5 μM for 8 h). *, P< 0.05 vs. LV-GFP-sh-con
group; #, P< 0.05 vs. LV-GFP-shGba #6 group; n=3.
Figure S4. C2-ceramide treatment conditions for maximal PPP2A activity. Optimal C2
concentration and application time (5 μM for 8 h) were determined according to the peak
increase in PPP2A activity. *P<0.05 vs. control group, #P<0.05 vs. other C2 treatment groups;
n=6.
Figure S5. The role of the macroautophagy pathway on SNCA accumulation. (A, B) The
efficiency of ATG5 knockdown was assessed 3 days after infection. The level of ATG5 protein
was reduced in cortical neurons infected with LV-cherry-shAtg5, compared to control neurons.
(C, D) As predicted, a reduction in LC3-II level was also observed in LV-cherry-shAtg5-infected
neurons, followed by the increase of SNCA, which was rescued by treatment with rapamycin
(40 nM for 6 h). *, P<0.05; **, P<0.01; ***, P<0.001 vs. LV-cherry-sh-con group; #, P< 0.05 vs.
LV-cherry-shAtg5 group; n=3.
Figure S6. SNCA overexpression leads to downregulation of GBA protein. GBA protein
expression, PPP2A activity, and the level of LC3-II were reduced in SK-N-SH neuroblastoma
cells transfected with pCMV-myc-SNCA (A to D) and cortical neurons infected with
LV-GFP-SNCA (E to H). *, P< 0.05; **, P< 0.01 vs. normal; n = 3.
Figure S7. SNCA accumulation contributes to neurodegeneration in GBA-deficient neurons.
(A) Autophagy level and GBA protein level in two transgenic mice overexpressing HsSNCA.
The SNCA level was significantly higher in cortex of transgenic mice (TG) than wild type (WT),
followed by decreased LC3-II and GBA protein level. (B, C) Decreased cell viability and
increased LDH release by GBA knockdown in cortical neurons, which was reversed by
treatment with C2 or rapamycin. **, P< 0.01 compared to LV-sh-con group, #, P< 0.05;
0.01 compared to LV-GFP-shGba #6 group; n = 6.
##,
P<
Figure S8. Loss of GBA function leads to PPP2A inactivation. Expression of PPP2A subunits
was assessed by western blotting in (A to D) SK-N-SH neuroblastoma cells transfected with
shGBA #9848-1 and (E to H) cortical neurons infected with LV-GFP-shGba #6. (B, F) PPP2R1
level was unaltered, and (C, G) PPP2R level and (D, H) PPP2C methylation were reduced in
GBA-deficient cells relative to control cells. *, P< 0.05, vs. normal; *, P< 0.05 vs.
LV-GFP-sh-con group; n = 3.